In response to environmental signals, a cell changes both its shape and its degree of attachment to a substratum. Changes in cell motility are brought about, in part, by rearrangements of the actin cytoskeleton. Changes in actin are associated with changes in cell morphology, growth, adhesion, and motility (Hall, A. (1994) Annu. Rev. Cell Biol. 10, 31-54; Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654; Vojtek, A. B. and Cooper, J. A. (1995) Cell 82, 527-529; Takai, Y., et al. (1995) TIBS 227-231.). In long bundles, F-actin supports filipodia, finger-like protrusions of the plasma membrane; as a meshwork, F-actin supports sheet-like protrusions of lamellipodia, called ruffles; in bundles coupled to attachment plaques, F-actin stress fibers exert force against a substratum (Zigmone (1996) Curr. Opin. Cell Biol. 8:66). This remodeling requires actin polymerization and depolymerization, which is orchestrated in part by members of the rho family of small GTPases (GTP-binding proteins), including rhoA, rac1, and cdc42 (Boguski and McCormick, (1993). Nature, 366:643-654; Hall, (1994). Annu. Rev. Cell Biol., 10:31-54; Ridley, (1995). Curr. Opon. Gen. Dev., 5:24-30; Zigmond, (1996). Curr. Opin. Cell Biol., 8:66-73). When active forms of the ras-like GTP-binding proteins are injected into cells, each of the family members induces unique morphological changes that involve rearrangement of F-actin. For example, in fibroblasts cdc42 regulates actin polymerization and focal complexes necessary for filopodia formation; rac mediates actin polymerization and focal complex assembly within lamellipodia and membrane ruffles; and rho induces actin stress fiber and focal adhesion (FA) complex formation (Nishiyama et al., (1994). Mol Cell. Biol., 14:2447-2456; Ridley, (1994). BioEssays, 16:321-327; Ridley and Hall, (1994). EMBO J., 13:2600-2610; Nobes and Hall, (1995). Cell, 81:53-62; Nobes et al., (1995). J. Cell Science, 108:225-233; Ridley et al., (1995). Mol. Cell. Biol., 15:1110-1122). In addition to these specific changes, rho family members share some of the same functions and there is cross-talk among members (Zigmone, supra). A hierarchical relationship exists among cdc42, rac, and rho, whereby cdc42 regulates rac activity and rac regulates rho activity, suggesting that these proteins may orchestrate the spatial and temporal changes in the actin cytoskeleton necessary for complex processes such as cell motility and cytokinesis (Ridley and Hall, (1992). Cell, 70:389-399; Chant and Stowers, (1995). Cell, 81:1-4; Nobes and Hall, (1995). Cell, 81:53-62; Lauffenburger and Horwitz, (1996). Cell, 84:359-369). Rho-like GTPases also play an essential role in cell cycle progression (Olson et al., (1995). Science, 269:1270-1272; Ridley, (1995). Curr. Opin. Gen. Dev., 5:24-30), ras-mediated cell transformation (Khosravi-Far et al., (1995). Mol. Cell. Biol., 15:6443-6453; Qiu et al., (1995). Proc. Natl. Acad. Sci. USA, 92:11781-11785), transcriptional regulation (Hill et al., (1995). Cell, 81:1159-1170), growth factor-induced arachidonic acid release and calcium influx (Peppelenbosch et al., (1995). Cell, 81:849-856; Peppelenbosch et al., (1996). J Biol. Chem., 271:7883-7886), and possibly HIV-1 replication (Lu et al., (1996). Curr. Biol., 6:1677-1684).
Rho-like GTPases function as molecular switches that are active when bound to GTP and inactive when bound to GDP (Boguski and McCormick, (1993)). Nature, 366:643-654). The activation state is positively regulated by guanine nucleotide exchange factors (GEFs) which promote the exchange of GDP for GTP, and negatively regulated by GTPase activating proteins (GAPs) (Boguski and McCormick, (1993). Nature, 366:643-654; Lamarche and Hall, (1994). TIG, 10:446-440; Overbeck et al., (1995). Mol. Repro. Dev., 42:468-476; Cerione and Zheng, (1996). Curr. Opin. Cell Biol., 8:216-222). In addition to GEFs and GAPs, the activation status of rho-like GTPases is controlled by GDP dissociation inhibitors and GDP dissociation stimulators (Boguski and McCormick, (1993). Nature, 366:643-654). About 20 GEFs for rho-like GTPases have been identified by sequence comparison (the Dbl homology (DH) GEF family), and a majority of these were shown to have GEF activity in vitro (Cerione and Zheng, (1996). Curr. Opin. Cell Biol., 8:216-222). Most of the DH GEFs were originally isolated as oncogenes including Dbl (Ron et al., 1988. EMBO J., 7:2465-2473; Hart et al., 1991. Nature, 354:311-314; Hart et al., (1994). J. Biol. Chem., 269:62-65), Ost (Horii et al., (1994). EMBO J., 13:4776-4786), and the invasion-inducing Tiam-1 oncogene (Habets et al., (1994). Cell, 77:537-549; Michiels et al., (1995). Nature, 375:338-340; van Leeuwen et al., (1995). Oncogene, 11:2215-2221). Upstream regulators of the rho/rac GEFs include growth factor receptors with protein tyrosine kinase activity (e.g., the insulin, EGF, and PDGF receptors), and seven transmembrane domain receptors coupled to heterotrimeric G proteins (e.g., the lysophosphatidic acid (LPA), bombesin, and bradykinin receptors) (Cerione and Zheng, (1996). Curr. Opin. Cell Biol., 8:216-222). In addition, growth factor-mediated activation of rho/rac GEF may in some cases involve phosphatidylinositol (PI)-3 kinase (Nobes et al., (1995). J. Cell Science, 108:225-233; Tsakiridis et al., (1996). J. Biol. Chem., 271:19664-19667).
A number of studies indicate that activated rho-like GTPases function as regulators of kinases. Rac and cdc42 were shown to activate members of the family of p21-activated serine/threonine kinases (PAKs) (Manser et al., (1994). Nature, 367:40-46; Bagrodia et al., (1995). J. Biol. Chem., 47:27995-27998; Knaus et al., (1995). Science, 269:221-223; Martin et al., (1995). E.M.B.O. J., 14:1970-1978; Frost et al., (1996). Mol. Cell. Biol., 16:3707-3713; Jakobi et al., (1996). J. Biol. Chem., 271:6206-6211). These kinases are homologous to the yeast STE20 kinase, which is involved in regulating a yeast MAP kinase cascade controlling mating pheromone response, polarity establishment, and filamentous growth of diploids (Ottilie et al., (1995). EMBO J., 14:5908-5918; Simon et al., (1995). Nature, 376:702-705; Stevenson et al., (1995). Genes Dev., 9:2949-2963). Rac and cdc42 also activate the mitogen-activated kinase (MAPK) family members Jun N-terminal kinase (JNK, also known as stress activated protein kinase (SAPK)) and p38 MAPK (Coso et al., (1995). Cell, 81:1137-1146.; Minden et al., (1995). Cell, 81:1147-1157; Pombo et al., (1995). Nature, 377:750-754; Vojtek and Cooper, (1995). Cell, 82:527-529; Zhang et al., (1995). J. Biol. Chem., 270:23934-23936), in addition activating the 70 kDa S6 kinase (Chou and Blenis, (1996). Cell, 85:573-583). Rho activates protein kinase N (PKN) (Amano et al., (1996). J. Biol. Chem., 271:20246-20249; Watanabe et al., (1996). Science, 271:645-648), p160.sup.ROCK kinase (Ishizaki et al., (1996). EMBO J., 15:1885-1893), and rho-kinase (Matsui et al., (1996). EMBO J., 15:2208-2216.). Rho-kinase was shown to phosphorylate the myosin light chain (MLC) (Amano et al., (1996). J. Biol. Chem., 271:20246-20249) and the myosin-binding subunit (MBS) of the myosin phosphatase, which results in the inactivation of myosin phosphatase and increased MLC phosphorylation (Kimura et al., (1996). Science, 273:245-248). Rho-kinase phosphorylates myosin light chain (MLC) and phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in non-muscle cells (Chrzanowska-Wodnicka and Burridge, (1996). J. Cell Biol., 133:1403-1415). Rho-like GTPases have also been shown to regulate PI 4-phosphate 5-kinase (PIP 5-kinase) (Chong et al., (1994). Cell, 79:507-513), PI 3-kinase (Zheng et al., (1994). J. Biol. Chem., 269:18727-18730; Tolias et al., (1995). J. Biol. Chem., 270:17656-17659; Bokoch et al., (1996). Biochem. J., 315:775-779), and phospholipase D (Malcolm et al., (1994). J. Biol. Chem., 269:25951-25954; Balboa and Insel, (1995). J. Biol. Chem., 270:29843-29847; Kwak et al., (1995). J. Biol. Chem., 270:27093-27098). Localized increases in PIP.sub.2 levels has been suggested to control actin polymerization and FA formation (Chong et al., (1994). Cell, 79:507-513; Hartwig et al., (1995). Cell, 82:643-653; Gilmore and Burridge, (1996). Nature, 381:531-534).
In addition to regulating kinases rho-like GTPases are involved in the regulation of other proteins, including the multicomponent NADPH oxidase (Diekmann et al., (1994). Science, 265:531-533; Knaus et al., (1995). Regulation of human leukocyte p21-activated kinases through G protein-coupled receptors. Science, 269:221-223), tubulin (Best et al., (1996). J. Biol. Chem., 271:3756-3762), and POR1 which is involved in membrane ruffling (Van Aelst et al., (1996). EMBO J., 15:3778-3786).
Proteins with GEF activity have also been implicated in cellular transformation. For example, several members of the Dbl family, which may function as GEFs for the rho-like proteins, have oncogenic activity (Adams et al., (1992) Oncogene 7:611; Miki et al (1993) Nature 362:462). Activated rac1 cooperates with a membrane-targeted form of raf (raf-CAAX) in oncogenic transformation (Qiu et al. (1995) Nature 374:457). In addition, rac and rho are essential for ras transformation of cells (Qiu, R. et al. (1995) Nature 374, 457-459; Khosravi-Far, R., et al. (1995) Mol. Cell. Biol. 15, 6443-6453). Cdc42, rho, and rac all appear to stimulate c-fos transcription (Hill, C. S., et al. (1995) Cell 81, 1159-1170), as well as cell cycle progression through G1 and subsequent DNA synthesis (Olson, M., et al. (1995) Science 269, 1270-1272). Rac is also involved in the activation of the NADPH oxidase complex in neutrophils (Segal and Abo (1993) Trends Biochem. Sci. 18:43).
Another protein thought to play a role in rearrangement of the actin cytoskeleton is the leukocyte common antigen related (LAR) transmembrane protein tyrosine phosphatase (PTPase). LAR is widely expressed and is comprised of a cell-adhesion-like extracellular region and two intracellular PTPase domains (Streuli, M., et al. (1992) EMBO J. 11, 897-907; Yu, Q., et al. (1992) Oncogene 7, 1051-1057; Fischer, E. H., et al. (1991) Science 253, 401-406; Mourey, R. J. and Dixon, J. E. (1994) Curr. Op. Gen. Dev. 4, 31-39). A role for LAR in regulating cell-matrix interactions was proposed, based on the colocalization of LAR with a coiled-coil protein, termed LAR interacting protein.1 (LIP.1) at the ends of FAs (Serra-Pages, C., et al. (1995) EMBO J. 14, 2827-2838). In addition LAR expression has been observed at regions of association between cells and basement membrane in various tissues (Streuli, M., et al. (1992) EMBO J. 11, 897-907).
Thus, certain biological functions such as growth, differentiation, and migration are tightly regulated by these signal transduction pathways within cells. Disregulation of normal activation pathways removes this tight control resulting in disease states, such as transformation. The development of agents capable of modulating ras-like GTP-binding proteins is clearly desirable, given the salient role of these molecules in regulating numerous aspects of cellular activation.